本文選題：紫花苜蓿 + 擬南芥��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2013年碩士論文

【摘要】：草甘膦（Glyphosate）商品名稱(chēng)為農(nóng)達(dá)，是一種廣譜高效的滅生性除草劑，在農(nóng)業(yè)生產(chǎn)中被廣泛應(yīng)用。紫花苜蓿（Medicago sativa L.）是一種多年生豆科飼草作物，有“牧草之王”的美譽(yù)，在我國(guó)種植廣泛。農(nóng)田雜草的滋生，不僅增加了成本投入，而且影響了牧草的產(chǎn)量和品質(zhì)。本研究將具有自主知識(shí)產(chǎn)權(quán)的抗草甘膦基因GR79Mt轉(zhuǎn)入擬南芥和苜蓿中，以期獲得具有草甘膦抗性的植株。 本實(shí)驗(yàn)得出如下結(jié)果： 1.建立了中苜6號(hào)組織培養(yǎng)再生體系。以下胚軸為外植體愈傷組織誘導(dǎo)率要高于以子葉為外植體的誘導(dǎo)率。在含2mg/L2,4-D和0.2mg/L KT的SH培養(yǎng)基上誘導(dǎo)愈傷組織效率最高，而分化培養(yǎng)基中僅含0.2mg/L的KT即可高效地誘導(dǎo)出胚狀體。同時(shí)，1g/L水解酪蛋白的加入，對(duì)胚狀體的產(chǎn)生和成熟有重要作用。胚狀體或小植株在不含植物激素的1/2MS培養(yǎng)基中誘導(dǎo)生根。 2.根據(jù)已知的抗草甘膦GR79Mt基因序列，在其前端加入葉綠體轉(zhuǎn)運(yùn)肽基因(CTP2)序列，由增強(qiáng)型啟動(dòng)子CAMV35S啟動(dòng)轉(zhuǎn)錄，豌豆rbcS E9基因的多聚腺嘌呤序列終止轉(zhuǎn)錄。遺傳元件構(gòu)建完成后人工合成序列，兩端加入PvuⅠ酶切位點(diǎn)。植物表達(dá)載體pCAMBIA2300用PvuⅠ酶切后留下原核表達(dá)框，與合成序列連接，成功的構(gòu)建了表達(dá)載體pCA-GM。 3.通過(guò)農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化擬南芥花序，將收獲的種子在含60mg/L草甘膦的MS培養(yǎng)基篩選，獲得T1代植株。提取其基因組DNA進(jìn)行PCR、RT-PCR驗(yàn)證，目的基因可在轉(zhuǎn)錄水平上表達(dá)，說(shuō)明目的基因已整合到擬南芥基因組上。 4.通過(guò)農(nóng)桿菌介導(dǎo)的雙邊界雙元載體pCA-GM，，轉(zhuǎn)化中苜6號(hào)愈傷組織。誘導(dǎo)培養(yǎng)基中含2mg/L2,4-D、0.2mg/L KT和60mg/L的草甘膦。誘導(dǎo)胚狀體時(shí)，KT濃度調(diào)整為0.4mg/L，草甘膦濃度增加至80mg/L。在無(wú)植物生長(zhǎng)調(diào)節(jié)劑的1/2MS培養(yǎng)基中誘導(dǎo)生根。最后獲得12株苜蓿再生植株，經(jīng)PCR驗(yàn)證兩株呈陽(yáng)性，初步表明目的基因已整合到苜�；蚪M中。
[Abstract]:Glyphosate (Glyphosate) is a broad-spectrum and high-efficient herbicide, which is widely used in agricultural production. Medicago sativa L. Is a perennial legume forage crop, with the "King of forage" reputation, widely planted in China. The breeding of weeds in farmland not only increases the cost input, but also affects the yield and quality of forage grass. In this study, glyphosate resistant gene GR79Mt with independent intellectual property rights was transferred into Arabidopsis thaliana and alfalfa in order to obtain glyphosate resistant plants. The results of this experiment are as follows: 1. The regeneration system of tissue culture of Zhongmu No.6 was established. The callus induction rate of cotyledons was higher than that of cotyledon explants. The callus induction efficiency was the highest on SH medium containing 2mg / L _ 2N _ 4-D and 0.2mg/L KT, but the embryoid was induced efficiently by KT containing only 0.2mg/L in the differentiation medium. At the same time, the addition of 1 g / L hydrolyzed casein plays an important role in the formation and maturation of embryoid. Embryoids or small plants were induced to take root in 1/2MS medium without plant hormones. 2. According to the known glyphosate-resistant GR79Mt gene sequence, the chloroplast transporter peptide gene (CTP2) sequence was added to the front of the gene, and the transcription was initiated by the enhanced promoter CAMV35S, and the polyadenine sequence of the pea rbcS E9 gene was terminated. After the construction of the genetic element, the synthetic sequence was constructed and the Pvu 鈪

本文編號(hào)：1938780